The dopamine (DA) transporter (DAT) controls DA homeostasis and neurotransmission by the active reuptake of synaptically released DA. The DAT is the major molecular target responsible for the rewarding properties and the abuse potential of amphetamine (AMPH) and cocaine. AMPH acts as a DAT substrate, promoting the reversal of DA transport, thereby resulting in DA efflux via DAT. This efflux leads to increased extracellular DA levels, an event of importance for the psychomotor stimulant properties of AMPHs. The N-terminus of the DAT is a structural domain that is critical for AMPH to cause DA efflux. We have shown that DAT N-terminus phosphorylation at the five most distal Ser is required for AMPH-induced DA efflux, but does not regulate DA uptake. Furthermore, our preliminary studies suggest that the DAT N-terminus interacts electrostatically with phosphatidylinositol-4,5-bisphosphate PIP2 (a key phospholipid enriched at the inner leaflet of the plasma membrane), and that this interaction impairs DA efflux, but not uptake. Our mechanistic hypothesis is that upon phosphorylation, the DAT N-terminus uncouples from PIP2 to disengage from the membrane. Both of these events are required to elicit the DA efflux produced by AMPH. We propose to test our hypothesis and its implications for the behavioral effects of AMPH in vivo, through the following specific aims: 1) To determine the nature of the interaction between DAT N-terminus and PIP2, and describe it in a structural context provided by computational modeling; 2) To determine the role of N-terminus phosphorylation in regulating how DAT and PIP2 interact, and its effect on DAT function; 3) To determine the role of DAT/PIP2 interactions in AMPH-induced behaviors in Drosophila melanogaster. In this animal model, we have established that locomotion is a DAT-dependent behavior and is stimulated by AMPH. Deletion of Drosophila DAT (dDAT) in DA neurons of flies inhibits AMPH-induced locomotion, an effect that is restored by the expression of the human DAT (hDAT) in these dDAT-deficient DA neurons. Using this strategy, we will translate in vivo our molecular findings from specific aims 1 and 2. This will allow us to understand how hDAT N-terminus phosphorylation and associations with PIP2 determine AMPH-induced behaviors. The long-term goals of this research are to understand how AMPH-induced DA efflux and its associated behaviors are dictated by the interactions of hDAT N-terminus with PIP2, and/or by phosphorylation of the N- terminus. By specifically impairing DA efflux, but not uptake, we will determine the contribution of DA efflux in AMPH behaviors. The intent is to uncover novel targets for the treatment of AMPH abuse. From a broad perspective of transporter biology, we will reveal how a DAT structural domain (the N-terminus) via its interactions with the plasma membrane dictates different aspects of transport function.